Regulatory T cells in human geohelminth infection suppress immune responses to BCG and Plasmodium falciparum

Chronic helminth infections induce T‐cell hyporesponsiveness, which may affect immune responses to other pathogens or to vaccines. This study investigates the influence of Treg activity on proliferation and cytokine responses to BCG and Plasmodium falciparum‐parasitized RBC in Indonesian schoolchildren. Geohelminth‐infected children's in vitro T‐cell proliferation to either BCG or pRBC was reduced compared to that of uninfected children. Although the frequency of CD4⁺CD25^hiFOXP3⁺ T cells was similar regardless of infection status, the suppressive activity differed between geohelminth‐infected and geohelminth‐uninfected groups: Ag‐specific proliferative responses increased upon CD4⁺CD25^hi T‐cell depletion in geohelminth‐infected subjects only. In addition, IFN‐γ production in response to both BCG and parasitized RBC was increased after removal of CD4⁺CD25^hi T cells. These data demonstrate that geohelminth‐associated Treg influence immune responses to bystander Ag of mycobacteria and plasmodia. Geohelminth‐induced immune modulation may have important consequences for co‐endemic infections and vaccine trials.     

Mycobacterium bovis BCG Vaccination Induces Divergent Proinflammatory or Regulatory T Cell Responses in Adults

Mycobacterium bovis bacillus Calmette-Guérin (BCG), the only currently available vaccine against tuberculosis, induces variable protection in adults. Immune correlates of protection are lacking, and analyses on cytokine-producing T cell subsets in protected versus unprotected cohorts have yielded inconsistent results. We studied the primary T cell response, both proinflammatory and regulatory T cell responses, induced by BCG vaccination in adults. Twelve healthy adult volunteers who were tuberculin skin test (TST) negative, QuantiFERON test (QFT) negative, and BCG naive were vaccinated with BCG and followed up prospectively. BCG vaccination induced an unexpectedly dichotomous immune response in this small, BCG-naive, young-adult cohort: BCG vaccination induced either gamma interferon-positive (IFN-γ⁺) interleukin 2-positive (IL-2⁺) tumor necrosis factor α-positive (TNF-α⁺) polyfunctional CD4⁺ T cells concurrent with CD4⁺ IL-17A⁺ and CD8⁺ IFN-γ⁺ T cells or, in contrast, virtually absent cytokine responses with induction of CD8⁺ regulatory T cells. Significant induction of polyfunctional CD4⁺ IFN-γ⁺ IL-2⁺ TNF-α⁺ T cells and IFN-γ production by peripheral blood mononuclear cells (PBMCs) was confined to individuals with strong immunization-induced local skin inflammation and increased serum C-reactive protein (CRP). Conversely, in individuals with mild inflammation, regulatory-like CD8⁺ T cells were uniquely induced. Thus, BCG vaccination either induced a broad proinflammatory T cell response with local inflammatory reactogenicity or, in contrast, a predominant CD8⁺ regulatory T cell response with mild local inflammation, poor cytokine induction, and absent polyfunctional CD4⁺ T cells. Further detailed fine mapping of the heterogeneous host response to BCG vaccination using classical and nonclassical immune markers will enhance our understanding of the mechanisms and determinants that underlie the induction of apparently opposite immune responses and how these impact the ability of BCG to induce protective immunity to TB.     

Stimulation of human peripheral blood mononuclear cells with live Mycobacterium bovis BCG activates cytolytic CD8+ T cells in vitro

Experimental data have shown that Mycobacterium tuberculosis can survive within the host cell and in doing so may release secreted antigen into the endogenous antigen-processing pathway. If mycobacterial antigen can gain access to MHC class I molecules then CD8⁺ T cells may play a role in host defence against M. tuberculosis infection. To identify whether there is a role for the CD8⁺ T cell in mycobacterial infection we have stimulated peripheral blood mononuclear cells (PBMC) from bacillus Calmette–Guérin (BCG) vaccinated individuals with live M. bovis BCG. The activation state of the T cells was established by staining for the interleukin-2 (IL-2) receptor (CD25), HLA-DR or the transferrin receptor (CD71). Using FACScan analysis we have shown that, in vitro, live M. bovis BCG activates significantly more CD8⁺ T cells in comparison to the soluble antigen purified protein derivative (PPD). In addition, live M. bovis BCG activates more CD8⁺ T cells than a non-viable preparation of the same M. bovis BCG following irradiation. The function of the activated CD8⁺ T cells was addressed using positively selected cells in a cytotoxic T-cell assay. CD8⁺ T cells isolated from a 7-day M. bovis BCG-stimulated PBMC culture were shown to be cytolytic against target cells infected with live M. bovis BCG, dead M. bovis BCG, and to a lesser extent, PPD. These results suggest that CD8⁺ T cells may be activated by stimulation with live mycobacteria, and that this subset can play a cytolytic role in the immune response to mycobacterial infections.     

The role of the alternative coreceptor GPR15 in SIV tropism for human cells

Many SIV isolates can employ the orphan receptor GPR15 as coreceptor for efficient entry into transfected cell lines, but the role of endogenously expressed GPR15 in SIV cell tropism is largely unclear. Here, we show that several human B and T cell lines express GPR15 on the cell surface, including the T/B cell hybrid cell line CEMx174, and that GPR15 expression is essential for SIV infection of CEMx174 cells. In addition, GPR15 expression was detected on subsets of primary human CD4⁺, CD8⁺ and CD19⁺ peripheral blood mononuclear cells (PBMCs), respectively. However, GPR15⁺ PBMCs were not efficiently infected by HIV and SIV, including cells from individuals homozygous for the defective Δ32 ccr5 allele. These results suggest that GPR15 is coexpressed with CD4 on PBMCs but that infection of CD4⁺, GPR15⁺ cells is not responsible for the well documented ability of SIV to infect CCR5⁻ blood cells.     

Programming cytomegalovirus as an HIV vaccine

The initial development of cytomegalovirus (CMV) as a vaccine vector for HIV/simian immunodeficiency virus (SIV) was predicated on its potential to pre-position high-frequency, effector-differentiated, CD8⁺ T cells in tissues for immediate immune interception of nascent primary infection. This goal was achieved and also led to the unexpected discoveries that non-human primate (NHP) CMVs can be programmed to differentially elicit CD8⁺ T cell responses that recognize viral peptides via classical MHC-Ia, and/or MHC-II, and/or MHC-E, and that MHC-E-restricted CD8⁺ T cell responses can uniquely mediate stringent arrest and subsequent clearance of highly pathogenic SIV, an unprecedented type of vaccine-mediated protection. These discoveries delineate CMV vector-elicited MHC-E-restricted CD8⁺ T cells as a functionally distinct T cell response with the potential for superior efficacy against HIV-1, and possibly other infectious agents or cancers.     

Mycobacterium chelonae sensitisation induces CD4+‐mediated cytotoxicity against BCG

Significant variability in efficacy of live Mycobacterium bovis BCG as a tuberculosis vaccine is observed globally. Effects of pre‐vaccination sensitisation to non‐tuberculous environmental mycobacteria (Env) are suspected to underlie this phenomenon, but the mechanisms remain unclear. We postulated that it could be due to Env‐specific T cells exerting cytotoxicity against BCG‐infected host cells. After murine sensitisation with heat‐killed antigens of different Env species, splenocytes from M. chelonae (CHE)‐sensitised mice exerted the strongest cytotoxicity against autologous BCG‐infected macrophages. This cytotoxicity was correlated with reduced BCG viability. The cytotoxicity was reduced by the depletion of CD4⁺, but not CD8⁺ or CD56⁺ cells, and CD4⁺ cells showed higher percentage of cytotoxicity than CD4^? cells, supporting a role for CD4⁺ cells in CHE‐induced, BCG‐specific cytotoxicity. Additionally, this cytotoxicity was IFN‐γ, perforin and FasL dependent. After CHE‐sensitisation and subsequent BCG intranasal infection, there was significant expansion of lung CD4⁺ cells, the main cell type producing IFN‐γ. This was associated with 2‐ and 6‐fold reductions in lung BCG counts 1 and 3 wk, respectively post‐ infection, relative to non‐sensitised mice. This is the first report describing cytotoxicity against BCG‐infected cells as a mechanism underlying the influence of Env sensitisation on subsequent BCG responses.     

Efficient Activation of Human T Cells of Both CD4 and CD8 Subsets by Urease-Deficient Recombinant Mycobacterium bovis BCG That Produced a Heat Shock Protein 70-M. tuberculosis-Derived Major Membrane Protein II Fusion Protein

For the purpose of obtaining Mycobacterium bovis bacillus Calmette-Guérin (BCG) capable of activating human naive T cells, urease-deficient BCG expressing a fusion protein composed of Mycobacterium tuberculosis-derived major membrane protein II (MMP-II) and heat shock protein 70 (HSP70) of BCG (BCG-DHTM) was produced. BCG-DHTM secreted the HSP70-MMP-II fusion protein and effectively activated human monocyte-derived dendritic cells (DCs) by inducing phenotypic changes and enhanced cytokine production. BCG-DHTM-infected DCs activated naive T cells of both CD4 and naive CD8 subsets, in an antigen (Ag)-dependent manner. The T cell activation induced by BCG-DHTM was inhibited by the pretreatment of DCs with chloroquine. The naive CD8⁺ T cell activation was mediated by the transporter associated with antigen presentation (TAP) and the proteosome-dependent cytosolic cross-priming pathway. Memory CD8⁺ T cells and perforin-producing effector CD8⁺ T cells were efficiently produced from the naive T cell population by BCG-DHTM stimulation. Single primary infection with BCG-DHTM in C57BL/6 mice efficiently produced T cells responsive to in vitro secondary stimulation with HSP70, MMP-II, and M. tuberculosis-derived cytosolic protein and inhibited the multiplication of subsequently aerosol-challenged M. tuberculosis more efficiently than did vector control BCG. These results indicate that the introduction of MMP-II and HSP70 into urease-deficient BCG may be useful for improving BCG for control of tuberculosis.     

Characterisation of simian immunodeficiency virus-infected cells in pigtail macaques

Defining which cells become infected with simian immunodeficiency virus (SIV) in vivo should assist in unravelling the pathogenesis of human immunodeficiency virus (HIV)/SIV infection. HIV/SIV infection of CD4⁺ T cells resulted in down-regulation of CD3 and CD4 surface molecules in vitro, however this phenomenon is poorly characterised in vivo. Intracellular SIV p27 was studied by flow cytometry in serial blood samples and lymph node samples during acute infection of 17 SIVmac-infected pigtail macaques. Two weeks after infection, a mean of 56±6.8% the p27⁺ cells were lymphocytes negative for surface CD4 and CD3, and indeed the highest proportion of SIV infected cells were found in the small subset of CD3^LoCD4⁻CD8⁻ lymphocytes, indicating that infection has lead to down-regulation of these markers in vivo. Furthermore, the relative amount of SIV p27 within lymphocytes (based of mean fluorescence intensity) was higher in CD3^LoCD4⁻ and CD3⁻ infected cells than in CD3⁺ or CD4⁺ p27⁺ populations, consistent with greater viral production in CD4⁺ T cells down-regulating CD3 and CD4 molecules. The CD3⁻CD4⁻ infected cells expressed T cell markers CD2 and CD5 and were negative for monocyte, NK and B cell markers. The majority of infected cells were CD28⁺CD95⁺ central memory T cells. Surprisingly, p27⁺ blood lymphocytes were mostly negative for activation markers CD25 and CD69, but most of the infected lymph nodes cells were activated. Our results characterise productively-infected macaque lymphocytes in vivo. The high proportion of SIV-infected lymphocytes that are CD3⁻CD4⁻ has important implications for the in vivo study of pathogenesis of SIV/HIV infections.     

Therapeutic envelope vaccination in combination with antiretroviral therapy temporarily rescues SIV‐specific CD4+ T‐cell‐dependent natural killer cell effector responses in chronically infected rhesus macaques

Natural killer (NK) cells are essential components of the immune system, and due to their rapid response potential, can have a great impact during early anti‐viral immune responses. We have previously shown that interleukin‐2‐dependent NK and CD4⁺ T‐cell co‐operative immune responses exist in long‐term simian immunodeficiency virus (SIV) ‐infected controlling macaques and can be rescued in SIV‐infected non‐controlling macaques by a short course of antiretroviral therapy (ART). Given that co‐operative responses may play an important role in disease prevention and therapeutic treatment, in the present study we sought to determine if these responses can be enhanced in chronically SIV‐infected macaques by vaccination with a single‐dose of envelope protein given during ART. To this end, we treated 14 chronically SIV‐infected macaques with ART for 11 weeks and gave 10 of these macaques a single intramuscular dose of SIV gp120 at week 9 of treatment. ART significantly decreased plasma and mucosal viral loads, increased the numbers of circulating CD4⁺ T cells in all macaques, and increased T‐cell‐dependent envelope‐ and gag‐specific interferon‐γ and tumour necrosis factor‐α production by circulatory CD56⁺ NK cells. The therapeutic envelope immunization resulted in higher envelope‐specific responses compared with those in macaques that received ART only. Functional T‐cell responses restored by ART and therapeutic Env immunization were correlated with transiently reduced plasma viraemia levels following ART release. Collectively our results indicate that SIV‐specific T‐cell‐dependent NK cell responses can be efficiently rescued by ART in chronically SIV‐infected macaques and that therapeutic immunization may be beneficial in previously vaccinated individuals.     

Exposure to human alveolar lining fluid enhances Mycobacterium bovis BCG vaccine efficacy against Mycobacterium tuberculosis infection in a CD8+ T-cell-dependent manner

Current tuberculosis (TB) treatments include chemotherapy and preventative vaccination with Mycobacterium bovis Bacillus Calmette-Guérin (BCG). In humans, however, BCG vaccination fails to fully protect against pulmonary TB. Few studies have considered the impact of the human lung mucosa (alveolar lining fluid (ALF)), which modifies the Mycobacterium tuberculosis (M.tb) cell wall, revealing alternate antigenic epitopes on the bacterium surface that alter its pathogenicity. We hypothesized that ALF-induced modification of BCG would induce better protection against aerosol infection with M.tb. Here we vaccinated mice with ALF-exposed BCG, mimicking the mycobacterial cell surface properties that would be present in the lung during M.tb infection. ALF-exposed BCG-vaccinated mice were more effective at reducing M.tb bacterial burden in the lung and spleen, and had reduced lung inflammation at late stages of M.tb infection. Improved BCG efficacy was associated with increased numbers of memory CD8⁺ T cells, and CD8⁺ T cells with the potential to produce interferon-γ in the lung in response to M.tb challenge. Depletion studies confirmed an essential role for CD8⁺ T cells in controlling M.tb bacterial burden. We conclude that ALF modifications to the M.tb cell wall in vivo are relevant in the context of vaccine design.     

Damage to the Blood-Brain Barrier during Experimental Cerebral Malaria Results from Synergistic Effects of CD8+ T Cells with Different Specificities

CD8⁺ T cells play a pathogenic role in the development of murine experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA (PbA) infection in C57BL/6 mice. Only a limited number of CD8⁺ epitopes have been described. Here, we report the identification of a new epitope from the bergheilysin protein recognized by PbA-specific CD8⁺ T cells. Induction and functionality of these specific CD8⁺ T cells were investigated in parallel with previously reported epitopes, using new tools such as tetramers and reporter cell lines that were developed for this study. We demonstrate that CD8⁺ T cells of diverse specificities induced during PbA infection share many characteristics. They express cytolytic markers (gamma interferon [IFN-γ], granzyme B) and chemokine receptors (CXCR3, CCR5) and damage the blood-brain barrier in vivo. Our earlier finding that brain microvessels in mice infected with PbA, but not with non-ECM-causing strains, cross-presented a shared epitope was generalizable to these additional epitopes. Suppressing the induction of specific CD8⁺ T cells through tolerization with a high-dose peptide injection was unable to confer protection against ECM, suggesting that CD8⁺ T cells of other specificities participate in this process. The tools that we developed can be used to further investigate the heterogeneity of CD8⁺ T cell responses that are involved in ECM.     

The Secreted Protein Rv1860 of Mycobacterium tuberculosis Stimulates Human Polyfunctional CD8+ T Cells

We previously reported that Rv1860 protein from Mycobacterium tuberculosis stimulated CD4⁺ and CD8⁺ T cells secreting gamma interferon (IFN-γ) in healthy purified protein derivative (PPD)-positive individuals and protected guinea pigs immunized with a DNA vaccine and a recombinant poxvirus expressing Rv1860 from a challenge with virulent M. tuberculosis. We now show Rv1860-specific polyfunctional T (PFT) cell responses in the blood of healthy latently M. tuberculosis-infected individuals dominated by CD8⁺ T cells, using a panel of 32 overlapping peptides spanning the length of Rv1860. Multiple subsets of CD8⁺ PFT cells were significantly more numerous in healthy latently infected volunteers (HV) than in tuberculosis (TB) patients (PAT). The responses of peripheral blood mononuclear cells (PBMC) from PAT to the peptides of Rv1860 were dominated by tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) secretions, the former coming predominantly from non-T cell sources. Notably, the pattern of the T cell response to Rv1860 was distinctly different from those of the widely studied M. tuberculosis antigens ESAT-6, CFP-10, Ag85A, and Ag85B, which elicited CD4⁺ T cell-dominated responses as previously reported in other cohorts. We further identified a peptide spanning amino acids 21 to 39 of the Rv1860 protein with the potential to distinguish latent TB infection from disease due to its ability to stimulate differential cytokine signatures in HV and PAT. We suggest that a TB vaccine carrying these and other CD8⁺ T-cell-stimulating antigens has the potential to prevent progression of latent M. tuberculosis infection to TB disease.     

MHC‐restricted Ag85B‐specific CD8+ T cells are enhanced by recombinant BCG prime and DNA boost immunization in mice

Despite efforts to develop effective treatments and vaccines, Mycobacterium tuberculosis (Mtb), particularly pulmonary Mtb, continues to provide major health challenges worldwide. To improve immunization against the persistent health challenge of Mtb infection, we have studied the CD8⁺ T cell response to Bacillus Calmette‐Guérin (BCG) and recombinant BCG (rBCG) in mice. Here, we generated CD8⁺ T cells with an rBCG‐based vaccine encoding the Ag85B protein of M. kansasii, termed rBCG‐Mkan85B, followed by boosting with plasmid DNA expressing the Ag85B gene (DNA‐Mkan85B). We identified two MHC‐I (H2‐K^d)‐restricted epitopes that induce cross‐reactive responses to Mtb and other related mycobacteria in both BALB/c (H2^d) and CB6F1 (H2^b/d) mice. The H2‐K^d‐restricted peptide epitopes elicited polyfunctional CD8⁺ T cell responses that were also highly cross‐reactive with those of other proteins of the Ag85 complex. Tetramer staining indicated that the two H2‐K^d‐restricted epitopes elicit distinct CD8⁺ T cell populations, a result explained by the X‐ray structure of the two peptide/H2‐K^d complexes. These results suggest that rBCG‐Mkan85B vector‐based immunization and DNA‐Mkan85B boost may enhance CD8⁺ T cell response to Mtb, and might help to overcome the limited effectiveness of the current BCG in eliciting tuberculosis immunity.     

Combined Env- and Gag-specific T cell responses in relation to programmed death-1 receptor and CD4+ T cell loss rates in human immunodeficiency virus-1 infection

Additional progression markers for human immunodeficiency virus (HIV) infection are warranted. In this study we related antigen-specific responses in CD4⁺ and CD8⁺ T cells to CD38, reflecting chronic immune activation, and to CD4⁺ T cell loss rates. Clones transiently expressing CD107a (CD8⁺) or CD154 (CD4⁺) in response to Gag, Env and Nef overlapping peptide pools were identified, along with their expression of the inhibitory programmed death-1 receptor (PD-1) in fresh peripheral blood mononuclear cells (PBMC) from 31 patients off antiretroviral treatment (ART). HIV-specific CD8⁺ T cell responses dominated over CD4⁺ T cell responses, and among CD8⁺ responses, Gag and Nef responses were higher than Env-responses (P < 0·01). PD-1 on CD8⁺ HIV-specific subsets was higher than CMV-specific CD8⁺ cells (P < 0·01), whereas PD-1 on HIV-specific CD4⁺ cells was similar to PD-1 on CMV-specific CD4⁺ cells. Gag and Env CD8⁺ responses correlated oppositely to the CD4 loss rate. Env/Gag CD8⁺ response ratios, independently of PD-1 levels, correlated more strongly to CD4 change rates (r = −0·50 to −0·77, P < 0·01) than the total number of Gag-specific CD8⁺ cells (r = 0·44–0·85, P ≤ 0·02). The Env/Gag ratio performed better than CD38 and HIV-RNA in logistic regression analysis predicting CD4 change rate as a measure of progression. In conclusion, HIV-specific CD8⁺CD107a⁺ Env/Gag response ratio was a stronger predictor for progression than CD38 and HIV-RNA. The Env/Gag ratio may reflect the balance between possibly beneficial (Gag) and detrimental (Env) CD8⁺ T cell responses and should be explored further as a progression marker.     

PTPN2 restrains CD8+ T cell responses after antigen cross-presentation for the maintenance of peripheral tolerance in mice

Antigen cross-presentation by dendritic cells is crucial for priming cytotoxic CD8⁺ T cells to invading pathogens and tumour antigens, as well as mediating peripheral tolerance to self-antigens. The protein tyrosine phosphatase N2 (PTPN2) attenuates T cell receptor (TCR) signalling and tunes CD8⁺ T cell responses in vivo. In this study we have examined the role of PTPN2 in the maintenance of peripheral tolerance after the cross-presentation of pancreatic β-cell antigens. The transfer of OVA-specific OT-I CD8⁺ T cells (C57BL/6) into RIP-mOVA recipients expressing OVA in pancreatic β-cells only results in islet destruction when OVA-specific CD4⁺ T cells are co-transferred. Herein we report that PTPN2-deficient OT-I CD8⁺ T cells transferred into RIP-mOVA recipients acquire CTL activity and result in β cell destruction and the development of diabetes in the absence of CD4⁺ help. These studies identify PTPN2 as a critical mediator of peripheral T cell tolerance limiting CD8⁺ T cell responses after the cross-presentation of self-antigens. Our findings reveal a mechanism by which PTPN2 SNPs might convert a tolerogenic CD8⁺ T cell response into one capable of causing the destruction of pancreatic β-cells. Moreover, our results provide insight into potential approaches for enhancing T cell-mediated immunity and/or T cell adoptive tumour immunotherapy.     

Accelerated induction of mycobacterial antigen-specific CD8+ T cells in the Mycobacterium tuberculosis-infected lung by subcutaneous vaccination with Mycobacterium bovis bacille Calmette–Guérin

Both CD4⁺ and CD8⁺ T cells are important in protection against Mycobacterium tuberculosis infection. To evaluate the effect of vaccination with Mycobacterium bovis bacille Calmette–Guérin (BCG) on the CD8⁺ T-cell response to pulmonary M. tuberculosis infection, we analyzed the kinetics of CD8⁺ T cells specific to the mycobacterial Mtb32a_309–318 epitope, which is shared by M. tuberculosis and M. bovis BCG, in the lung of mice infected with M. tuberculosis. The CD8⁺ T cells were detected by staining lymphocytes with pentameric major histocompatibility complex (MHC) class I H-2D^b–Mtb32a_209–318 peptide complex and were analysed by flow cytometry. Mtb32a-specific CD8⁺ T cells became detectable on day 14, and reached a plateau on day 21, in the lung of M. tuberculosis-infected unvaccinated mice. Subcutaneous vaccination with M. bovis BCG in the footpads induced Mtb32a-specific CD8⁺ T cells in the draining lymph nodes (LNs) on day 7 and their numbers further increased on day 14. When M. bovis BCG-vaccinated mice were exposed to pulmonaryinfection with M. tuberculosis 4 weeks after vaccination, the Mtb32a-specific CD8⁺ T cells in the infected lung became detectable on day 7 and reached a plateau on day 14, which was 1 week earlier than in the unvaccinated mice. The pulmonary CD8⁺ T cells from the BCG-vaccinated M. tuberculosis-infected mice produced interferon-γ in response to Mtb32a_209–318 peptide on day 7 of the infection, whereas those of unvaccinated mice did not. The results demonstrate that induction of mycobacterial antigen-specific protective CD8⁺ T cells in the M. tuberculosis-infected lung is accelerated by subcutaneous vaccination with M. bovis BCG.     

CD4+ Th1 cells promote CD8+ Tc1 cell survival, memory response, tumor localization and therapy by targeted delivery of interleukin 2 via acquired pMHC I complexes

The cooperative role of CD4⁺ helper T (Th) cells has been reported for CD8⁺ cytotoxic T (Tc) cells in tumor eradication. However, its molecular mechanisms have not been well elucidated. We have recently demonstrated that CD4⁺ Th cells can acquire major histocompatibility complex/peptide I (pMHC I) complexes and costimulatory molecules by dendritic cell (DC) activation, and further stimulate naïve CD8⁺ T cell proliferation and activation. In this study, we used CD4⁺ Th1 and CD8⁺ Tc1 cells derived from ovalbumin (OVA)-specific T cell receptor (TCR) transgenic OT II and OT I mice to study CD4⁺ Th1 cell''s help effects on active CD8⁺ Tc1 cells and the molecular mechanisms involved in CD8⁺ Tc1-cell immunotherapy of OVA-expressing EG7 tumors. Our data showed that CD4⁺ Th1 cells with acquired pMHC I by OVA-pulsed DC (DC_OVA) stimulation are capable of prolonging survival and reducing apoptosis formation of active CD8⁺ Tc1 cells in vitro, and promoting CD8⁺ Tc1 cell tumor localization and memory responses in vivo by 3-folds. A combined adoptive T-cell therapy of CD8⁺ Tc1 with CD4⁺ Th1 cells resulted in regression of well-established EG7 tumors (5 mm in diameter) in all 10/10 mice. The CD4⁺ Th1’s help effect is mediated via the helper cytokine IL-2 specifically targeted to CD8⁺ Tc1 cells in vivo by acquired pMHC I complexes. Taken together, these results will have important implications for designing adoptive T-cell immunotherapy protocols in treatment of solid tumors.     

Heat‐Shock Protein gp96 Enhances T Cell Responses and Protective Potential to Bacillus Calmette‐Guérin Vaccine

The commonly used Bacillus Calmette‐Guérin (BCG) vaccine only induces moderate T cell responses and is less effective in protecting against pulmonary tuberculosis (TB) in adults and ageing populations. Thus, developing new TB vaccine candidates is an important strategy against the spread of Mycobacterium tuberculosis. Here, we demonstrated that immunization with heat‐shock protein gp96 as an adjuvant led to a significantly increased CD4⁺ and CD8⁺ T cell response to a BCG vaccine. Secretion of the Th1‐type cytokines was increased by splenocytes from gp96‐immunized mice. In addition, adding gp96 as an adjuvant effectively improved the protection against intravenous challenge with Mycobacterium bovis BCG in mice. Our study reveals the novel property of gp96 in boosting the vaccine‐specific T cell response and its potential use as an adjuvant for BCG vaccines against mycobacterial infection.     

Live attenuated Salmonella Typhimurium with monophosphoryl lipid A retains ability to induce T-cell and humoral immune responses against heterologous polysaccharide of Shigella flexneri 2a

Shigella flexneri 2a (Sf2a) is one of the most frequently isolated Shigella strains that causes the endemic shigellosis in developing countries. In this study, we used recombinant attenuated Salmonella vaccine (RASV) strains to deliver Sf2a O-antigen and characterized the immune responses induced by the vectored O-antigen. First, we identified genes sufficient for biosynthesis of Sf2a O-antigen. A plasmid containing the identified genes was then introduced into the RASV strains, which were manipulated to produce only the heterologous O-antigen and modified lipid A. After oral immunization of mice, we demonstrated that RASV strains could induce potent humoral immune responses as well as robust CD4⁺ T-cell responses against Sf2a Lipopolysaccharide (LPS) and protect mice against virulent Sf2a challenge. The induced serum antibodies mediated high levels of Shigella-specific serum bactericidal activity and C3 deposition. Moreover, the IgG⁺ B220^low/int B_M cell and T follicular helper (Tfh) cell responses could also be triggered effectively. The live attenuated Salmonella with the modified lipid A delivering Sf2a O-antigen polysaccharide showed the same ability to induce immune responses against Sf2a LPS as the strain with the original lipid A. These findings underscore the potential of RASV delivered Sf2a O-antigen for induction of robust CD4⁺ T-cell and IgG responses and warrant further studies toward the development of Shigella vaccine candidates with RASV strains.